Introduction to Gene Expression, & Microarray Technology
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Transcript of Introduction to Gene Expression, & Microarray Technology
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Introduction to Gene Expression,
& Microarray Technology
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Gene expression
• Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product.
• These products are often proteins
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Also known as DNA Chip
• Allows simultaneous measurement of the level of transcription for every gene in a genome (gene expression)
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• Are small, solid supports onto which the sequences or subsequences from thousands of different genes are attached.
• The supports are usually glass microscope slides, or silicon chips or nylon membranes. The DNA is printed, spotted, or actually synthesized directly onto the support.
• The spots can be DNA, cDNA, or oligonucleotides
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• It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, each containing specific DNA sequence.
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DNA microarrays are created by spotting every gene in a genome onto a glass microscope slide.
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Modified from http://darwin.bio.uci.edu/~faculty/wagner/array2.html
Each spot represents different gene/clone
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Why Are Microarrays Important?
• Microarrays are a significant advance both because they may contain a very large number of genes and because of their small size.
• Microarrays are therefore useful when one wants to survey a large number of genes quickly or when the sample to be studied is small.
• .
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The process
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Sample preparation
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• The two samples to be compared .
• In this example treated sample (case) and untreated sample (control).
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DNA probe
• A short sequence of DNA labelled that is used for the detection of a complementary nucleotide sequence.
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Hybridization
cover
slip
Hybridize for
5-12 hours
Binding of cDNA target samples to cDNA probes on the slide
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DNA microarrays: step by step
• Production of DNA probes
• Printing or “spotting” Printing or “spotting”
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Ngai Lab arrayer , UC Berkeley
The arrayer
Print-tip head
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• Once extracted, the mRNAs need to be labelled with fluorescent markers1 so that they can be detected later, on the surface of the micorarray.
• mRNA of the control cells is usually labelled with green fluorescent marker, and mRNA of the cells under study with red fluorescent marker.
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• the control mRNA (labelled green) is mixed with the test mRNA (labelled red).
• The mixture is then flooded over the surface of a slide, which is then incubated at 42°C,
• After 12 hours, the microarray is washed .
• The microarray is now ready for scanning.
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Labeled DNA hybridizes to corresponding DNA/gene
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ScanningDetector
PMT
Image
Duplicate spots
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• But wait a minute there are not just red and green dots there are yellow dots as well!
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• This can easily be explained.
• It is clear that the red spots contain mRNA from cancer cells and green spots mRNA from noncancerous control cells.
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• yellow! Remember mRNA hybridizes with its complementary DNA and one spot on the microarray represents billions of copies of DNA from ONE gene.
• In other words, when a spot is yellow, there are equals amounts of mRNA of the gene found in cancerous and control cells.
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• And black means that there is no mRNA of that gene either in the control or cancerous cells.
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RGB overlay of Cy3 and Cy5 images
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